Endometrial thickness (EMT) minimal stimulation(left; E2 781 pg/mL; EMT 4.0 mm) versus frozen embryo transfer (right; E2 296 pg/mL; EMT 8.8 mm) in the same patient
20.5.2 The Effect of Clomiphene Citrate on Histomorphology
Underlying the effects of clomiphenecitrate on endometrial thickness is its effects on endometrial differentiation at the cellular level. In endometrial samples obtained from patients undergoing minimal stimulation vs conventional stimulation at the time of oocyte retrieval, we noted stark differences. After minimal stimulation, small undifferentiated glands are dispersed in a sea of polygonal stromal cells. The glands remain narrow and tubular, as expected during the early-to-mid-proliferative phase. In contrast, large tortuous glands of secretory epithelium are the major component of endometrium after conventional stimulation (Fig. 20.2). Furthermore, histomorphometric analyses reveal an increased number of glands per cross-sectional area, along with decreased gland diameter and epithelial height suggesting decreased secretory differentiationwith minimal stimulation (Table 20.1).
Fig. 20.2
H&E staining of endometrial biopsysamples from minimal stimulation (left) vs conventional stimulation (right)
Table 20.1
Morphometric analysis of endometrial biopsy samples
Minimal stimulation
Conventional stimulation
P
Number of glands per mm2
68 ± 21.2
33 ± 5.1
0.003
Gland volume fraction (%)
19.9 ± 2.4
41.5 ± 6.4
<0.0001
Average max gland diameter (μm)
64.7 ± 5.9
159.5 ± 25.2
0.001
Average gland height (μm)
17.2 ± 1.9
21.2 ± 1.4
0.002
20.5.3 The Effect of Clomiphene Citrate on Gene Expression
We also observed profound differencesin endometrial gene expression between minimal and conventional stimulation. Specifically, 3.4% (723 of 20,764 genes) are significantly differentially expressed. Surprisingly, RNA sequencing does not reveal significant changes in the estrogen receptor or its co-regulators nor in classic proliferation-associated genes in the endometrium such as cyclinA and cMyc or antiproliferative genes such as p27Kip1. Perhaps, one of the most interesting pathways differentially expressed in the endometrium is that of WNT/β- catenin signaling which is known to be involved in endometrial proliferation. WNT7A is expressed by the luminal epithelium and is a diffusible factor that triggers cell proliferation [12]. Acting on the underlying stroma, WNT7A binds to the receptor Frizzled that phosphorylates the intracytoplasmic protein Dishevelled. When phosphorylated, Dishevelled inactivates glycogen synthase kinase β, turning off the breakdown of β-catenin by ubiquitination. The accumulation of β-catenin induces cell proliferation associated with endometrial growth.
Several WNT inhibitory genes are differentially expressed in minimal stimulation including NOTUM, WISE/SOST, WIF-1, SFRP1, and SFRP4. Secreted Frizzled-related proteins (sFRPs) antagonize Wnt signaling at the receptor level. Overexpression of sFRP4 and treatment with recombinant sFRPP4 protein inhibits endometrial cancer cellgrowth in vitro [13]. In endometrial samples comparing minimal stimulation to conventional stimulation, stromal-derived sFRP1 and sFRP4 were increased dramatically in endometrium obtained from clomiphene citrate-treated cycles. Immunofluorescence localized sFRPs to the endometrial stroma with notable accumulation directly underlying the glands, perhaps indicating paracrine signaling between the stroma and glands (Fig. 20.3). This is consistent with the current understanding that estrogen-induced stromal factors are required for epithelial proliferation. Estradiol increases epithelial cell proliferation when cocultured with stroma, but it does not increase proliferation in epithelial cellscultured in the absence of stromal cells.
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